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You searched for: EV190096 (EV-TRACK ID)

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Experiment number
  • If needed, multiple experiments were identified in a single publication based on differing sample types, separation protocols and/or vesicle types of interest.
Species
  • Species of origin of the EVs.
Separation protocol
  • Gives a short, non-chronological overview of the different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Details EV-TRACK code Experiment nr. Species Sample type Separation protocol First author Year EV-METRIC
EV190096 1/2 Bos taurus skim milk acetic acid treatment
DG
(d)(U)C
Filtration
Mukhopadhya, Anindya 2021 100%

Study summary

Full title
All authors
Anindya Mukhopadhya, Jessie Santoro, Barry Moran, Zivile Useckaite, Lorraine O'Driscoll
Journal
Food Chem.
Abstract
Many infants are fed infant milk formula (IMF). However, IMF production from skim milk (SM) involves (show more...)Many infants are fed infant milk formula (IMF). However, IMF production from skim milk (SM) involves harsh treatment. So, we hypothesised that the quantity and/or quality of extracellular vesicles (EVs) in IMF may be reduced. Thus, firstly, we aimed to optimise separation of EVs from IMF and SM and, secondly, we aimed to compare the EV isolates from these two sources. Prior to EV isolation, abundant casein micelles of similar sizes to EVs were removed by treating milk samples with either acetic acid or hydrochloric acid. Samples progressed to differential ultracentrifugation (DUC) or gradient ultracentrifugation (GUC). EV characterisation included BCA, SDS-PAGE, nanoparticle tracking (NTA), electron microscopy (TEM), immunoblotting, and imaging flow cytometry (IFCM). Reduced EV concentrations were found in IMF. SM-derived EVs were intact, while IMF contained disrupted EV-like structures. EV biomarkers were more abundant with isolates from SM, indicating EV proteins in IMF are compromised. Altogether, a suitable method combining acid pre-treatment with GUC for EV separation from milk products was developed. EVs appear to be substantially compromised in IMF compared to SM. (hide)
EV-METRIC
100% (50th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
skim milk
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
acetic acid treatment
DG
(d)(U)C
Filtration
Protein markers
EV: TSG101/ CD63/ CD81/ HLADR/ ADAM10/ CD9
non-EV: Actinin4
Proteomics
no
EV density (g/ml)
1.15
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Bos taurus
Sample Type
skim milk
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Equal to or above 150,000 g
Between 50,000 g and 100,000 g
Pelleting performed
No
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
5
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
17
Sample volume (mL)
2.33
Orientation
Bottom-up
Rotor type
Type 70.1Ti
Speed (g)
186000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
9
Pelleting: duration (min)
90
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
110000
Filtration steps
0.45µm > x > 0.22µm,
Other
Name other separation method
acetic acid treatment
Other
Name other separation method
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD63/ TSG101
Not detected EV-associated proteins
Detected contaminants
Not detected contaminants
Actinin4
Flow cytometry
Type of Flow cytometry
AMNIS ImageStreamX Mark II Flow Cytometer
Calibration bead size
none
Antibody details provided?
No
Detected EV-associated proteins
CD63/ CD9/ CD81/ ADAM10/ HLADR
Not detected EV-associated proteins
Detected contaminants
Not detected contaminants
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
160
EV concentration
Yes
EV190096 2/2 Bos taurus powdered infant milk formula acetic acid treatment
DG
(d)(U)C
Filtration
Mukhopadhya, Anindya 2021 100%

Study summary

Full title
All authors
Anindya Mukhopadhya, Jessie Santoro, Barry Moran, Zivile Useckaite, Lorraine O'Driscoll
Journal
Food Chem.
Abstract
Many infants are fed infant milk formula (IMF). However, IMF production from skim milk (SM) involves (show more...)Many infants are fed infant milk formula (IMF). However, IMF production from skim milk (SM) involves harsh treatment. So, we hypothesised that the quantity and/or quality of extracellular vesicles (EVs) in IMF may be reduced. Thus, firstly, we aimed to optimise separation of EVs from IMF and SM and, secondly, we aimed to compare the EV isolates from these two sources. Prior to EV isolation, abundant casein micelles of similar sizes to EVs were removed by treating milk samples with either acetic acid or hydrochloric acid. Samples progressed to differential ultracentrifugation (DUC) or gradient ultracentrifugation (GUC). EV characterisation included BCA, SDS-PAGE, nanoparticle tracking (NTA), electron microscopy (TEM), immunoblotting, and imaging flow cytometry (IFCM). Reduced EV concentrations were found in IMF. SM-derived EVs were intact, while IMF contained disrupted EV-like structures. EV biomarkers were more abundant with isolates from SM, indicating EV proteins in IMF are compromised. Altogether, a suitable method combining acid pre-treatment with GUC for EV separation from milk products was developed. EVs appear to be substantially compromised in IMF compared to SM. (hide)
EV-METRIC
100% (50th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
powdered infant milk formula
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
acetic acid treatment
DG
(d)(U)C
Filtration
Protein markers
EV: TSG101/ CD63/ CD81/ HLADR/ ADAM10/ CD9
non-EV: Actinin4
Proteomics
no
EV density (g/ml)
1.15
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Bos taurus
Sample Type
powdered infant milk formula
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Equal to or above 150,000 g
Between 50,000 g and 100,000 g
Pelleting performed
No
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
5
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
17
Sample volume (mL)
2.33
Orientation
Bottom-up
Rotor type
Type 70.1Ti
Speed (g)
186000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
9
Pelleting: duration (min)
90
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
110000
Filtration steps
0.45µm > x > 0.22µm,
Other
Name other separation method
acetic acid treatment
Other
Name other separation method
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD63/ TSG101
Not detected EV-associated proteins
Detected contaminants
Not detected contaminants
Actinin4
Flow cytometry
Type of Flow cytometry
AMNIS ImageStreamX Mark II Flow Cytometer
Calibration bead size
none
Antibody details provided?
No
Detected EV-associated proteins
CD63/ CD9/ CD81/ ADAM10/ HLADR
Not detected EV-associated proteins
Detected contaminants
Not detected contaminants
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
175
EV concentration
Yes
1 - 2 of 2
  • CM = Commercial method
  • dUC = differential ultracentrifugation
  • DG = density gradient
  • UF = ultrafiltration
  • SEC = size-exclusion chromatography
EV-TRACK ID
EV190096
species
Bos taurus
sample type
skim milk
powdered
infant milk formula
condition
Control condition
Control condition
separation protocol
acetic acid treatment
DG
(d)(U)C
Filtration
acetic acid treatment
DG
(d)(U)C
Filtration
Exp. nr.
1
2
EV-METRIC %
100
100